Apparatus and method for machining microchamber for cell culture

ABSTRACT

This invention relates to a method and apparatus for process cell cultures. The apparatus comprises a micro-chamber comprising no more than one absorption layer and at least one gel layer, in this order, laminated on a transparent base plate having no conspicuous absorbency in visible and infrared regions, and at least one light source. The absorption layer has absorbency in visible and infrared regions, and the gel-like material is a substance which has a gel dissolution temperature of 100 degree C. or less, solates when heated and is in a gel state at room temperature and has absorbency for a specific wave length of visible and infrared regions. The light source is monochromatic light in the specific wave length. The light source is disposed such that it irradiates on the absorption layer and/or gel layer, with the exception that when no absorption layer is provided, at least two layers each composed of a gel-like material are laminated on the transparent base plate.

FIELD OF THE INVENTION

The present invention relates to a micro-chamber processing apparatusand a micro-chamber processing method useful for the culture of cells.

PRIOR ART

Up to date, for the observation of a change in state of cells and aresponse to chemical agents, etc. of cells, it is common to observe theaverage value of some values of a cell population on the assumption thatit is a property of one cell. However, actually, it is seldom that cellssynchronize in their cell cycles in a group of them, but each cellrather develops a protein in a different cycle. In order to solve theseproblems, a technique such as a synchronous culture process, etc. hasbeen developed. However, because cultured cells are not derived from onecompletely identical cell, there was a possibility that a difference indevelopment of a protein is caused by a difference in gene of each cellderived before culture. Actually, when analyzing the results ofresponses to irritation, it was difficult to clarify whether itsfluctuation derives from that of general responses possessed by a cell'sreaction mechanism itself or whether it is derived from a difference incell (that is, a difference in gene information). For the same reasons,with respect to cell lines it was difficult to clarify whether thereproducibility of responses to irritation fluctuates due to adifference in gene of each cell because it is generally not culturedfrom one completely identical cell. Further, from the fact that thereare two types of irritation (signal) to cells, i.e., one being given bythe amounts of signal substance, nutriment and dissolved gas containedin a solution in the circumference of a cell, and the other beingprovided by the physical contact between cells, it was the circumstancethat it is difficult to judge its fluctuation.

On the other hand, heretofore, when cells are to be observed in a studyfield of biotechnology, it is common either to observe them by removinga portion of a cell group cultured in a large culture vessel and settingit on a microscope, or to conduct the observation with a microscope byenclosing the entire microscope with a plastic container to control thetemperature and then placing another small container within theenclosure under the control of carbon dioxide concentration andhumidity. Then, it is designed to exchange the used culture solutionwith a new culture solution while culturing cells whereby the solutionconditions are maintained constant. For example, there are a process ofmaintaining nutrient conditions constant by means of a mechanism whereina circulating pump operates upward and downward the level of a culturemedium relative to the surface of a base material between a level higherthan the upper end edge of the base material and a level lower than thelower end edge thereof in such a manner that when it decreases to thelower level, a culture medium is fed, while when it increases to thehigher level, a culture medium is discharged (Japanese PatentApplication Public Disclosure (Kokai) Hei 10-191961), and a process ofmaintaining the nutrient conditions of a culture vessel constant byinserting in a culture vessel one end of each of an inlet tube forintroducing a new culture medium into the culture vessel, an outlet tubefor discharging a culture medium from the culture vessel and a gas tubefor communicating a gas portion of the culture vessel with a pump,wherein the inlet tube, the outlet tube and the gas tube are provided ontheir respective conduit line with a filter for preventing the intrusionof bacilli into the culture vessel (Japanese Patent Application PublicDisclosure (Kokai) Hei 8-172956). However, in either of theseinventions, a technique is not known wherein cells are cultured whilecontrolling the solution environment of a cell to be cultured and thephysical contact between cells.

Accordingly, we solved these problems, and invented a technique ofselecting a specific new one cell only and culturing the one cell as acell line, a technique wherein, when observing cells, the solutionenvironment conditions of the cells are controlled and the cellconcentration in the vessel is maintained constant, and a technique ofobserving the culture while specifying interacting cells (PatentApplication Public Disclosure (Japanese Patent Application PublicDisclosure (Kokai) 2002-153260). Further, we invented a micro-chamberfor cell culture wherein the shape of a cell culture vessel can befreely varied while culturing cells in the region irradiated and heatedwith a convergent light (Patent Application No. 2002-245904).

PROBLEMS TO BE SOLVED BY THE INVENTION

In the preparation of micro-chambers for cell culture, it is possible tomake electrode array base plates and physical barriers taking advantageof a micro-processing technique having been developed by utilizing asemi-conductor fabrication technique. In order to process and modifybase plates, however, it was necessary to repeat complicated steps suchas light exposure, etching, etc. in a clean room, etc. to previouslyincorporate therein a shape and a pattern prior to beginning of theculture of cells. Accordingly, it was difficult to simply alter thestructure immediately before the commence of culture of cells, toprocess the shape during the culture of nerve cells, to alter the finestructure depending on the behavior of cells, and also to conduct thecontinuous processing while confirming the processing position withvisual observation during processing.

By using, as a structural material for the base plate in the culture ofnerve cells, a soft material (a gel-like material) which can be easilydissolved by heating with a convergent light, the present invention aimsat providing a micro-chamber array technique by which it is possible to,simply and freely, add an etching process depending on the observationon the state of cells, and also it is possible to conduct the continuousprocessing while confirming the shape when processing, which weredifficult in conventional micro-fabrication technique using, as the rawmaterial, hard materials such as glasses, silicones, etc.

MEANS TO SOLVE THE SUBJECT

We have now found that when a layer composed of a gel-like materialhaving absorbency at a specific wave length of visible or infraredregions is formed on a base plate and this specific absorption wavelength is then irradiated on the resulting layer with the use of a lightsource of a monochromatic light, preferably a laser, whereby a chamberof a desired shape is formed in the layer composed of a gel-likematerial, then the resulting chamber is very suitable for cell culture,and attained the present invention.

The micro-chambers formed according to the processing apparatus ormethod of the present invention have advantages that (1) themicro-processing is possible immediately before the commence of cellculture or during the culture of cells, (2) it is possible to controlthe expansion of a physical or biochemical neurite taking advantage ofthe inertness to cells and the non-adhesiveness to cells of a gel-likematerial such as agarose, etc., (3) the etching is possible with aresolution power of approximately 1 μm; It is easy to form a complicatedshape such as a tunnel-type channel, etc., which was formed onlyaccording to a complicated process in prior arts, (4) micro-structurescan be easily fabricated only with a convergent light-type heatingapparatus without necessity of using expensive equipments such as aclean room, a mask aligner, a dry etching apparatus, etc., and like.

That is, the present invention is a micro-chamber processing apparatusfor cell culture comprising a micro-chamber and at least one lightsource, wherein said micro-chamber comprises no more than one absorptionlayer and at least one gel layer composed of a gel-like material(hereinafter called “gel layer”) or at least two gel layers when noabsorption layer is provided, in this order, laminated on a transparentbase plate having no conspicuous absorbency in visible and infraredregions, said absorption layer has absorbency in visible or infraredregion, said gel-like material is in a gel state at room temperature,which has a gel dissolution temperature of 100 degree C. or less andsolates when heated, said gel-like material has absorbency at a specificwave length of visible or infrared region, said light source has amonochromatic light at said specific wave length, and said light sourceis disposed such that it irradiates on said absorption layer and/or saidgel layer.

Further, the present invention is a method for processing micro-chambersfor cell culture which comprises the steps of:

preparing a micro-chamber comprises no more than one absorption layerand at least one gel layer composed of a gel-like material or at leasttwo gel layers when no absorption layer is provided, in this order,laminated on a transparent base plate having no conspicuous absorbencyin visible and infrared regions, said absorption layer has absorbency invisible or infrared region, said gel-like material is in a gel state atroom temperature, which has a gel dissolution temperature of 100 degreeC. or less and solates when heated, said gel-like material hasabsorbency at a specific wave length of visible or infrared region, and

irradiating at least one visible or infrared monochromatic light on theabsorption layer or the gel layer composed in the micro-chamber to formtherein a gel-free region of a desired shape.

Further, the present invention is a method for culturing cell whichcomprises, in addition to the method as in above, a step of injecting acell and its culture solution into a formed region free from a gelsubstance.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows one embodiment of a micro-chamber processing apparatusaccording to the present invention, wherein reference numerals indicatethose parts as follows; 100: a micro-chamber for cell culture, 101: abase plate, 102: an absorption layer, 103: a gel layer, 104: a visiblelight for optical observation, 105, 106: a light source of a specificwave length, 107, 114: an optical lens, 108: an objective lens, 109: aconvergent light of a specific wave length, 111,112: a mirror refractinga light of a specific wave length, 113: a mirror, and 115: a camera forobservation.

FIG. 2 illustrates a step of processing a desired shape in amicro-chamber. Reference alphabets a through c indicate a procedure offorming a tunnel, and d through f indicate a procedure of forming anupward opened hole, wherein reference numerals 204, 214: a convergentlight, 205, 215: a focal point of a convergent light, 206,216: amovement direction of a convergent light, 207: a diffusion direction ofa solated material, 208: a tunnel, and 217: a hole.

FIG. 3 shows microphotographs illustrating one embodiment of amicro-chamber processing method. Rows show the results of differentlaser strengths (93 mW, 108 mW, 120 mW), wherein the upper rows showphase-contrast microscopic images, the middle rows show top surfaceimages obtained by a confocal scanning microscope, and the lower rowsshow cross section images obtained by a confocal scanning microscope,wherein reference numeral 301 indicates a hole.

FIG. 4 shows microphotographs illustrating one embodiment of amicro-chamber processing method. Reference alphabets a through c aremicrophotographs as seen from the top surface, and d is a confocalscanning microscopic image showing the cross section of a base plate atstage c, wherein reference numerals 401 and 402 indicate a hole and atunnel, respectively.

FIG. 5 shows microphotographs illustrating one embodiment of amicro-chamber processing method, wherein reference numerals indicatethose parts as follows; 501: a base plate, 502, 503: a gel layer havinga different absorption wave length or gel dissolution temperature, 504,514: a convergent light, 505: a focal point of the convergent light,506: a movement direction of the convergent light, and 507: a diffusiondirection of a solated material.

EMBODIMENTS OF THE INVENTION

First, we will describe the constitution of a micro-chamber according tothe present invention.

It is desirable that the transparent base plate is a material which hasno conspicuous absorbency in visible and infrared regions and isoptically transparent for the light of a wave length selected forprocessing. It is preferable that this base plate has a relatively smallabsorption of less 0.1% as compared with that of the absorption layer asdescribed hereinafter for all wave lengths of visible and infraredregions used in the processing apparatus of the present invention. Moreparticularly, it is possible to use glasses such as borosilicate glass,quartz glass, etc., plastics such as polystyrene, etc., solid baseplates such as a silicone base plate, etc. and high molecular materialssuch as agarose, etc.

On this base plate is disposed an absorption layer having absorbency invisible and infrared regions. It is preferable that this absorptionlayer is a film formed of Cr or a metal oxide such as aluminum oxide,etc. In general, these films have an even absorption over the entirewave length of visible and infrared regions. However, there areabsorption and scattering peaks depending on the wave length of a lightand the film thickness such as Fabry-Perot. It, therefore, is betterthat it is thinner than the wave length of a light used. It ispreferable that it has an absorption of 1,000 times or more that of alight of a layer containing a gel-like material as described hereinafterfor the wave length used. For example, for a wave length of 1064 nm, thedeposited Cr layer of 5 nm has an absorption of 10% or more relative toirradiated light. The absorption of agarose is 0.01% or less. Thisabsorption layer can be also omitted.

Further, the base plate per se or the base plate having an absorptionlayer thereon can be treated with a collagen molecule or polylysine. Thesurface per se of the glass base plate can be made water-penetrative byoxygen ashing. For example, the surface of an absorption layer such as adeposited layer of chromium, etc. can be subjected to a silane-makingtreatment, on which is applied and fixed a cell absorption element suchas collagen, etc. Then, the processing can be conducted so that thecells can be stably adhered on the bottom of a hole. The conditions ofsuch surface treatments can be determined according to necessitydepending on a cell to be cultured and its object.

On this absorption layer is disposed at least one gel layer havingabsorbency for a specific wave length of visible and infrared regions,which material is a substance which has a gel dissolution temperature of100 degree C. or less preferably 45 degree C. or less, solates whenheated and is in a gel state at room temperature. The micro-chamber ofthe present invention features using a plurality of layers including anabsorption layer. Therefore, when it has no absorption layer, at leasttwo layers each composed of a gel-like material are laminated on thebase plate.

This gel-like material is a substance which undergoes a change in phaseof from a sol into a gel or from a gel into a sol in its solution whenheated or cooled, wherein this sol—gel change can irreversibly takeplace at a specific temperature of from 0 to 100 degree C. Thissubstance assumes a molecular structure of the random coil type in itsheated solution. When this solution is cooled, a portion of thesubstance assumes a helical structure so that it produces a network. Asa result, it is believed that this substance loses finally its fluidityso that it gels. When continuously cooled, this gel network increaseswith time so that it forms a stronger gel.

Such substances include straight chain polymers obtained by thepurification of biogenic materials such as, for example, collagen,agarose, agaropectin, galactose, anhydrogalactose, galacturonic acid andits methyl ester, etc. However, it is also possible to use synthetichigh molecules having the above functions. Particularly, it is believedthat it is optimum to use agarose because it has no adhesiveness tocells and is not signal substance against cells so that it is nontoxicto cells, and thus has little affection on culture test data.

This substance is dissolved in water or a buffer, etc. in aconcentration of generally from 0.2 to 10% depending on use applicationswhereby a gel-like material is formed.

This gel-like material can be a combination of gel-like materials havingeach a different gel dissolution temperature. For example, although theyare different depending on the chain length, etc. of purified molecules,in general, collagen has a gelation temperature of from 15 to 20 degreeC. and a gel dissolution temperature of 20 to 30 degree C., and agaroseand agaropectin have each a gelation temperature of from 30 to 40 degreeC. and a gel dissolution temperature of 85 degree C., respectively. Forgalactose and anhydrogalactose, the gelation temperature is from 30 to75 degree C. and the gel dissolution temperature is from 5 to 10 degreeC. higher than the gelation initiation temperature. For galacturonicacid and its methyl ester, the gelation temperature is from 60 to 80degree C. and the gel dissolution temperature is from 60 to 80 degree C.under the conditions of a sugar level of 65 degree or more and a pH of3.5, and the gelation temperature is from 30 to 40 degree C. and the geldissolution temperature is from 30 to 40 degree C. in the presence of acalcium ion.

Further, a substance (dye, etc.) absorbing infrared lights or visiblelights can be incorporated in this gel-like material so that it hasabsorbency for a specific wave length.

This gel-like material has an Abs of generally less than 0.01 for anoptical path length of 1 cm with respect to a wave length for which ithas no absorbency.

The thickness of the layer of this gel-like material can be determinedat need according to its object. In general, it is on the order of from100 nm to 2 nm.

When this gel-like material is used in the form of a multi-layerstructure, the layers each composed of a gel-like material may have eacha different absorption wave length, and also may have each a differentdissolution temperature. Further, these layers may be laminated in sucha manner that the gel dissolution temperature gradually increases towardabove on a base plate.

The micro-chamber of the present invention may either one which has oneabsorption layer and one gel layer laminated on a base plate, or onewhich has two layers each composed of a gel-like material laminated on abase plate and has no absorption layer thereon. Three or more materialshaving each a different melting point can be also laminated thereon.Further, the micro-chamber can be three-dimensionally divided into somezones so that it has a region of a different melting point and a regionof a different absorbency. It is possible to stepwise select adissolution region by controlling the type and strength of a heated andfocused light. More particularly, it can be realized by laminatingthereon low melting agaroses having each a different melting point.However, it is also possible to use different materials such as agarose,plastics, etc.

The light source used in the processing apparatus of the presentinvention emits a monochromatic light in visible and infrared regions.The light source can be used singly or in a combination of two or more.It is preferable that the light source is a laser. These lasers includeNd: YAG laser (1064 nm), Raman Fiber laser (1480 nm), titanium sapphirelaser (variable in 500-1100 nm), Alexandrite laser (variable in 700-818nm), color center laser (variable in 800-4,000 nm), OPO laser (variablein 400-800 nm), etc.

Such light sources are disposed in such a manner that they irradiate onan absorption layer and/or a gel layer. Particularly, it is preferablethat they are disposed such that they can focus and irradiate a light oneither of an absorption layer and/or a gel layer.

Further, this light source can be composed of two or more light sourceshaving each a different wave length, at least one wave length of whichmay be an absorption wave length for either gel layer. Alternatively, itcan consist of one light source, the wave length of which may be anabsorption wave length for either gel layer.

The processing apparatus may further have an instrument means,preferably, an optical microscope, for confirming the position of alight radiated from a light source during irradiation. By thedisposition of such an instrument means, the processing shape orprocessing stage can be visually confirmed optically with the use of aweak observation light.

In the processing apparatus having the above constitution, amonochromatic light is irradiated on an absorption layer or a gel layer.When it is irradiated on an absorption layer, a portion of a gel layeris locally dissolved by the heat generated in the absorption layer. Onthe other hand, when a light of an absorption wave length for a gel-likematerial is irradiated on this gel-like material, the gel-like materialis locally dissolved so that the material itself diffuses into thelayer. Then, the resulting space is filled with a water or buffercontained in the gel layer. By moving the position of such a lightirradiation according to necessity, a water-or buffer-filled space of adesired shape can be formed within a gel layer. The shape of such aspace is not particularly restricted to any form, but a cylindrical orrectangular body having a hole of from 2 μm to 1 mm in diameter, apassageway having a diameter of 2 μm to 1 mm and a length of from 2 μmto 1 mm, and the like can be formed.

In such a manner, a desired shape can be formed in a gel layer. Anoutward opened hole can be formed in an inner space, or a pipe can beinserted from outside into an inner space, whereby a cell or its culturesolution is injected into or discharged from its space.

Further, the gel layer can be covered over its outer surface with anoptically transparent semipermeable membrane such as cellulose, etc.whereby the contamination with extraneous microbes, etc. is preventedand also it is possible to prevent cells from escaping out from thehole. Then, for example, when the gel layer is agarose and thesemipermeable membrane is cellulose, a portion each of sugar chains ofthe both is ring-opened so as to modify avidin and biotin which haveeach an amino end in the —CHO residue whereby the semipermeable membranecan be bonded to the gel layer through an avidin-biotin bond. When it isnecessary to cycle a culture solution in the culture of cells, a gellayer is covered with an optically transparent container of a shapecovering over the entire layer. Thus, a culture solution can beintroduced into the container through a tube, and a waste liquid of theculture solution can be recovered from another tube communicating withthe container.

Now, we will describe an embodiment each of a micro-chamber processingapparatus, a processing method and a micro-chamber formed therebyaccording to the present invention. The present invention is not to berestricted to the following embodiments so that various embodiments arepossible.

FIG. 1 shows one embodiment of the micro-chamber processing apparatusesof the present invention. In a micro-chamber for cell culture 100according to the present invention, an absorption layer (a film layer)102 having optical absorbency such as a deposited layer of chromium,etc. is disposed on an optically transparent base plate 101 such as aslide glass, etc. For the observation with a transmitted light, it isdesirable that the absorption layer 102 has a film thickness of theorder that it does not entirely absorb a light and that it is uniformlythin. For example, when the absorption layer is a chromium deposit, ithas a film thickness of 50 Å wherein the transmitted light isapproximately 70% in a visible region. Then, on the absorption layer 102is laminated a layer 103 composed of a gel-like material such asagarose, etc., which is optically transparent, has a low melting pointand is free of toxicity to cells, etc. In the layer 103 are then formeda plurality of holes for the introduction of a sample such as cells,etc. according to a process described hereinafter. Thus, a specific cellcan be cultured in their respective hole.

The apparatus of FIG. 1 has a means for irradiating two or moreconvergent lights each of a different wave length through an objectivelens on a layer 103 composed of a gel-like material to thereby processthe shape of the layer 103. More particularly, for example, with respectto the light irradiated from a light source 105 which generates amonochromatic light of 1064 nm having no absorbency for water, theposition of a focal point at which it passes through an objective lens108 can be controlled by adjusting the position of a lens 107. Theposition of this focal point can be adjusted by the visual confirmationby means of an optical microscopic instrument means. Further, withrespect to the light irradiated from a light source 106 which generatesa monochromatic light of 1480 nm having absorbency for water, theposition of a focal point within a micro-chamber can be likewisecontrolled by adjusting the position of the lens 107. Thesemonochromatic lights can be introduced into the optical paths of theobjective lens by means of mirrors 111 and 112, respectively.

Further, in the apparatus of the present invention, the image observedby a visible light source 104 can be focused on the light-receivingsurface of a camera 115 by means of a lens 114 in order to confirm aposition to be processed by a convergent light of the specific wavelength.

FIG. 2 shows a step of processing, by the combination of two convergentlights each of a different wave length, a desired shape in amicro-chamber comprising one absorption layer and thereon one layer (forexample, 200 μm in thickness) composed of a gel-like material disposedon a base plate.

FIG. 2 a to c show a procedure of forming a tunnel 208 by irradiating aconvergent light of, for example, 1064 nm on an absorption layer. First,a convergent light 204 of 1064 nm is absorbed in an absorption layer 202absorbing a light of this wave length in the vicinity of a focal point205 so that the resulting heat dissolves a portion of a layer 203composed of a gel-like material. This gel-like material itself diffusesinto the layer 203, and instead the space thus formed is filled with awater or buffer contained in the layer 203. Then, when the position ofthis focal point 205 is moved to a direction of the arrow 206 (FIG. 2b), a tunnel 208 filled with water or a buffer is formed, with themovement of the focal point, in the layer 203 composed of a gel-likematerial as shown in FIG. 2 c.

As shown in FIG. 2 d to f, when a monochromatic convergent light 214 of1480 nm is irradiated on a layer 213 composed of a gel-like material,with respect to a layer 213 composed of a water-containing material suchas agarose, etc. all of the material are heated and dissolved on anoptical path 214 as water absorbs a convergent light of 1480 nm. As aresult, an upward opened hole is formed in the layer 213 composed of agel-like material (FIG. 2 d). A cell or its culture solution can beinjected or discharged through such an upward opened hole. Further, asshown in FIG. 2, when this focal point 215 is moved to a direction ofthe arrow 216, the shape of the tunnel is thereby enlarged (FIG. 2 f).

FIG. 3 shows microphotographs of the holes thus formed. FIG. 3illustrates a case where holes are formed by irradiating a convergentlight of 1480 nm for 30 seconds on a base plate having agarose coatedthereon. From this figure, it can be confirmed that holes are formedalong the optical path in an agarose layer of 200 μm in thickness.

FIG. 4 shows one example of processing procedures using two irradiationlights each of a different wave length. For example, by stepwise using amonochromatic light of 1064 nm and a monochromatic light of 1480 nm,holes as well as a tunnel for connecting these holes can be formed. FIG.4 a shows a photograph of a base plate before the irradiation of alight, and FIG. 4 b shows holes 401 formed with a monochromatic laserlight of 1480 nm according to the procedure illustrated in FIG. 3. Aftertwo holes are formed, as shown in FIG. 4 c and d the absorption layer isirradiated with a wave length of 1064 nm for which agarose has noabsorbency (according to the same procedure as that of FIG. 2 a-c) toform a tunnel 402 on the bottom of an agarose layer whereby two holescan be connected to each other.

FIG. 5 shows a process of forming a complicated shape by laminating on abase plate 501 two layers 502 and 503 each composed of a gal-likematerial having a different absorption wave length and then using twoconvergent lights 504 and 514 to selectively dissolve a layer 502 onlyand a layer 503 only, respectively. First, a wave length absorbed in thelayer 502 only is used as shown in Fi.5 a., and the layer 502 only isprocessed as shown in FIG. 5 b. Second, as shown in FIG. 5 c, the layer503 is processed with the use of a wave length absorbed in the layer503. Thus, it is possible to process complicated shapes.

It is also possible to form similar shapes by laminating on a base plate501 two layers 502 (low gel dissolution temperature) and 503 (high geldissolution temperature) which are each composed of a gel-like materialhaving a different gel dissolution temperature, and varying itsirradiation time with the use of a light source of one wave length.

ADVANTAGES OF THE INVENTION

As discussed in detail, according to the present invention it ispossible to vary the shape of a culture vessel in response to a cultureprocess while culturing bio-cells, etc., which was considered impossibleup to date. Further, it is possible to form a structure by using acombination of a plurality of lights each having a different wave lengthto locally dissolve a substance in a region less than that of a light.

1. A micro-chamber processing apparatus for cell culture comprising amicro-chamber and at least one light source, wherein said micro-chambercomprises at least two gel layers each composed of a gel materiallaminated on a transparent base plate having no conspicuous absorbencyin visible and infrared regions, said gel material is in a gel state atroom temperature, which has a gel dissolution temperature of 100 degreeC. or less and solates when heated, said gel material has absorbency ata specific wave length of visible or infrared region, said light sourcehas a monochromatic light at said specific wave length, and said lightsource is disposed such that it irradiates on said gel layers, whereinthe at least two gel layers each have a different absorption wave lengthor a different dissolution temperature.
 2. The apparatus as in claim 1further comprising a measuring means for confirming the position of alight irradiated from a light source during irradiation.
 3. Theapparatus as in claim 1 wherein the light source is disposed such thatit can focus and irradiate a light on either an absorption layer or agel layer.
 4. The apparatus as in claim 1 wherein the light sourcecomprises two or more light sources having each a different wave length,at least one wave length of which is an absorption wave length of eithergel layer.
 5. The apparatus as in claim 1 wherein the light sourcecomprises one light source, a wave length of which is an absorption wavelength of either gel layer.
 6. The apparatus as in claim 1 wherein noabsorption layer is provided.
 7. The apparatus as in claim 2 wherein thelight source is disposed such that it can focus and irradiate a light oneither of an absorption layer or a gel layer.
 8. The apparatus as inclaim 2 wherein the light source comprises two or more light sourceshaving each a different wave length, at least one wave length of whichis an absorption wave length of either gel layer.
 9. The apparatus as inclaim 2 wherein the light source comprises one light source, a wavelength of which is an absorption wave length of either gel layer. 10.The apparatus as in claim 2 wherein no absorption layer is provided. 11.The apparatus as in claim 7 wherein the light source comprises two ormore light sources having each a different wave length, at least onewave length of which is an absorption wave length of either gel layer.12. The apparatus as in claim 11 wherein the light source comprises onelight source, a wave length of which is an absorption wave length ofeither gel layer.
 13. The apparatus as in claim 12 wherein no absorptionlayer is provided.